Accomplishments and discoveries from VGN students and faculty

The Laboratory of Keith Mintz, PhD at UVM, with the VGN Proteomics Core, Identifies Proteins Necessary for Fimbrae Secretion and Biofilm Development in the Oral Pathogen Aggregatibacter actinomycetemcomitans


The cell envelope of Gram-negative bacteria is an essential structure required for cellular maintenance, protection from environmental stressors, and interactions with other microorganisms or eukaryotic tissues. Three physiochemically distinct but contiguous compartments compose this structure: the inner membrane, a phospholipid bilayer adjacent to the cytosol; the periplasm, a viscous aqueous compartment containing the peptidoglycan cell wall; and the outer membrane, an asymmetric bilayer composed of lipids and lipopolysaccharide exposed to the extracellular environment. Each compartment contains conserved proteins of general cellular functions and proteins exclusively associated with the specific organism.


Standing Left to Right: Yan Xing, David Danforth, Kenneth Smith, Keith Mintz
Kneeling Left to Right : Richard Voogt, Thomas Freeman

The loss of a conserved inner membrane protein, morphogenesis protein C (MorC) from the oral pathogen Aggregatibacter actinomycetemcomitans, dramatically changes the morphology and physiological properties of the outer membrane of the bacterium. To elucidate the role(s) of MorC in maintaining membrane morphology and function, we, in collaboration with the VGN Proteomics Facility, under took a proteomic approach to quantify differences in the membrane proteome of the wild type and isogenic morC mutant strains. Membrane proteins were extracted, differentially labeled and a multidimensional protein identification technique (MudPIT), utilizing ion exchange chromatography coupled with analysis by mass spectrometry, was employed. 665 proteins were identified from the wild type strain including surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple macromolecular transporters, and components of the type I, II and V secretion systems. 107 proteins with unknown function were associated with the cell envelope.


Fimbriae

Surprisingly, only 12 proteins were found in lesser (10) or greater abundance in the membrane preparation of the morC mutant strain compared with the wild-type strain. One protein (TadB) was associated with the secretion of fimbriae, hair-like protein projections required for biofilm formation. Subsequent investigation into the fimbriae production demonstrated that MorC influences fimbriae formation and altered the microcolony formation required for biofilm development in the oral pathogen A. actinomycetemcomitans.

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